CGI-58 knockdown in mice causes hepatic steatosis but prevents diet-induced obesity and glucose intolerance.

Mutations of Comparative Gene Identification-58 (CGI-58) in humans cause triglyceride (TG) accumulation in multiple tissues. Mice genetically lacking CGI-58 die shortly after birth due to a skin barrier defect. To study the role of CGI-58 in integrated lipid and energy metabolism, we utilized antisense oligonucleotides (ASOs) to inhibit CGI-58 expression in adult mice. Treatment with two distinct CGI-58-targeting ASOs resulted in ∼80–95% knockdown of CGI-58 protein expression in both liver and white adipose tissue. In chow-fed mice, ASO-mediated depletion of CGI-58 did not alter weight gain, plasma TG, or plasma glucose, yet raised hepatic TG levels ∼4-fold. When challenged with a high-fat diet (HFD), CGI-58 ASO-treated mice were protected against diet-induced obesity, but their hepatic contents of TG, diacylglycerols, and ceramides were all elevated, and intriguingly, their hepatic phosphatidylglycerol content was increased by 10-fold. These hepatic lipid alterations were associated with significant decreases in hepatic TG hydrolase activity, hepatic lipoprotein-TG secretion, and plasma concentrations of ketones, nonesterified fatty acids, and insulin. Additionally, HFD-fed CGI-58 ASO-treated mice were more glucose tolerant and insulin sensitive. Collectively, this work demonstrates that CGI-58 plays a critical role in limiting hepatic steatosis and maintaining hepatic glycerophospholipid homeostasis and has unmasked an unexpected role for CGI-58 in promoting HFD-induced obesity and insulin resistance.


Mice and ASO treatment
Male C57BL/6N mice were obtained from Harlan (Indianapolis, IN) at 3-4 weeks of age. At 8 weeks of age, mice were either maintained on standard rodent chow diet (Prolab RMH 3000) or switched to a synthetic HFD. The HFD was prepared by our institutional diet core and contains ‫ف‬ 45% of energy as lard and ‫ف‬ 0.015% (w/w) cholesterol. In conjunction with diet feeding, mice received biweekly intraperitoneal injections of either saline, a nontargeting control ASO (Control), or one of two independent ASOs targeting CGI-58 (CGI-58 ␣ ; or CGI-58 ␤ ) for 8 weeks. The 20-mer phosphorothioate ASOs were designed to contain 2'-0-methoxyethyl groups at positions 1 to 5 and 15 to 20, and were synthesized, screened, and purifi ed as described previously ( 23 ) by ISIS Pharmaceuticals (Carlsbad, CA). Following 8 weeks of dietary induction, all experimental animals were euthanized following a 4 h fast during the light cycle. Mice were maintained in a specifi c pathogen-free animal facility, and all experimental protocols were approved by the Institutional Animal Care and Use Committee at the Wake Forest University School of Medicine.

Plasma biochemistries
Unless otherwise stated, plasma samples were collected by submandibular vein puncture following a 4 h fast during the light cycle. For fasted and fed plasma samples, blood was collected by submandibular vein puncture at 9:00 AM in ad libitum fed mice (fed) or in mice fasted during the dark cycle for 12 h (9:00 PM-9:00 AM). Detailed description of plasma lipid and lipoprotein analyses has been previously described ( 24 ). Plasma ␤ -hydroxybutyrate (Stanbio Laboratory, Boerne, TX), nonesterifi ed fatty acids (NEFA-HR; Wako Diagnostics, Richmond, VA), aspartate aminotransferase, and alanine aminotransferase (Point Scientifi c, Inc. Canton, MI) were measured using commercially available kits. Plasma glucose levels were measured using a glucometer (Ascensia Countour, Bayer). Plasma insulin levels were measured by ELISA (Crystal Chem, Inc., Downers Grove, IL).

Hepatic lipid analyses
Extraction of liver lipids and enzymatic quantifi cation of total TG, cholesteryl esters (CEs), free cholesterol (FC), and PL were performed as previously described ( 24 ). Total liver NEFA was measured enzymatically (NEFA-HR; Wako Diagnostics) in detergent (1% Triton-X-100) solubilized liver extracts. Hepatic contents of diacylglycerol, ceramide, long-chain fatty acyl-CoA (LCCoA) and their molecular species were analyzed by liquid chromatography/tandem mass spectrometry as previously described ( 25,26 ). Hepatic glycerophospholipid analyses were conducted using multi-dimensional mass spectrometry-based shotgun lipidomics as previously described ( 27,28 ). Analyses were performed on a QqQ mass spectrometer (Thermo Fisher TSQ Quantum Ultra, San Jose, CA) equipped with an automated nanospray apparatus (i.e., Nanomate HD, Advion Bioscience Ltd., Ithaca, NY) and operated with an Xcalibur software system as previously described ( 29 ). multiple tissues including liver ( 1 ). All NLSD patients develop nonalcoholic fatty liver disease ( 1 ). A subset of patients with NLSD display thickened, scaly, dry skin known as ichthyosis ( 1 ). In the 1970s Chanarin et al. ( 2 ) and Dorfman et al. ( 3 ) reported some of these early cases and in recognition of their work, NLSD with ichthyosis was given the name Chanarin-Dorfman syndrome (CDS). In 2001, it was discovered that mutations in CGI-58 (also known as ␣ / ␤ hydrolase domain-containing protein-5, Abhd5) cause CDS ( 4 ). However, nearly a decade after the causal link between CGI-58 and CDS was established, we still do not completely understand how CGI-58 functions to prevent CDS.
Although the prevailing theory is that CGI-58 regulates cellular TG content by serving as a coactivator of ATGL ( 14,17,20 ), there have been two in vitro studies showing that CGI-58 is an acyl-CoA-dependent lysophosphatidic acid acyltransferase (LPAAT) that prefers arachidonoyl-CoA and oleoyl-CoA as substrates ( 21,22 ). Although the in vivo signifi cance of this in vitro fi nding remains to be explored, CGI-58 mutations causing CDS were shown to have normal LPAAT activity ( 21 ), arguing against a role of defective LPAAT activity of CGI-58 in CDS development.
Collectively, cell-based and in vitro studies support a critical role of CGI-58 in controlling TG hydrolysis. Recently, total body CGI-58 knockout mice were generated ( 11 ). Unfortunately, these mice exhibit neonatal lethality due to a defective skin barrier function, making it diffi cult to study the role of CGI-58 in integrated lipid and energy metabolism. To circumvent this limitation we have utilized antisense oligonucleotides (ASOs) to inhibit the expression of CGI-58 in adult mice. Results from these studies demonstrate that CGI-58 limits hepatic steatosis by regulating hepatic TG and PL metabolism, and unexpectedly link CGI-58 to high-fat diet (HFD)-induced obesity and insulin resistance. mRNA level for each gene represents the amount relative to that in the control ASO-treated group, which was arbitrarily standardized to 1. Primers used for qPCR are available on request.

Glucose homeostasis
Intraperitoneal glucose tolerance tests and insulin tolerance tests were performed as previously described ( 24 ) in mice treated with diet and ASO for 6 or 7 weeks, respectively.

Statistical analysis
Data are expressed as the mean ± SEM. All data were analyzed using either a one-way or two-way ANOVA followed by Student's t -tests for post hoc analysis. All analyses were performed using JMP version 5.0.12 (SAS Institute; Cary, NC) software.

ASO-mediated knockdown of CGI-58 prevents HFD-induced obesity
CGI-58 serves as a coactivator for ATGL-mediated TG hydrolysis in vitro ( 14 ). ATGL knockout mice exhibit mild obesity ( 34 ). Given this, one would assume that CGI-58 defi ciency would phenocopy the mild obesity seen in ATGL knockout mice. To examine this possibility, we treated adult mice with two distinct CGI-58-targeting ASOs, and this treatment resulted in ‫ف‬ 80-95% knockdown of CGI-58 mRNA and protein expression in white adipose tissue and liver ( Figs. 1A , B, 2A , B ). The most effective CGI-58 ASO, CGI-58 ␤ , also caused substantial protein knockdown in brown adipose tissue ( ‫ف‬ 50%), heart ( ‫ف‬ 80%), skin ( ‫ف‬ 70%), and spleen ( ‫ف‬ 50%) (supplementary Fig. I). CGI-58 ␣ ASO also knocked down CGI-58 protein expression in spleen by ‫ف‬ 60% (supplementary Fig. I). In contrast, levels of CGI-58 were not affected by ASO treatment in brain, lung, or skeletal muscle (supplementary Fig. I). In chow-fed mice, CGI-58 knockdown did not signifi cantly alter body weight ( Fig. 1C ), yet both CGI-58 ASOs promoted a ‫ف‬ 50% decrease in epididymal fat pad weight compared with control mice ( Fig. 1D ). When challenged with a HFD, the saline and control ASO-treated mice gained in excess of 10 g in body weight over the 8 week feeding period, whereas mice treated with CGI-58 ASOs were completely resistant to HFD-induced obesity ( Fig. 1C ). In agreement, CGI-58 ASO-treated HFD-fed mice had a ‫ف‬ 50% decrease in epididymal fat pad weight compared with control mice ( Fig.  1D ). Additionally, CGI-58 ASO-treated mice consumed the same amount of food over the 8 week feeding period (data not shown), likely indicating that CGI-58 ASO treatment promotes an increase in energy expenditure.

ASO-mediated knockdown of CGI-58 causes severe hepatic steatosis
Patients affected with CDS and CGI-58 knockout mice exhibit marked hepatic steatosis ( 1,11 ). ASO treatment was extremely effective in diminishing the hepatic expression of CGI-58, with the CGI-58 ␤ ASO resulting in >98% knockdown at the mRNA and protein level ( Fig. 2A, B ). Knockdown of CGI-58 signifi cantly decreased hepatic TG hydrolase activity on both diets ( Fig. 2C ), which was

Determination of hepatic lipid secretion rates
The chow-fed mice underwent 4 weeks of ASO treatment. Recirculating isolated liver perfusions were performed in fed mice as previously described ( 30 ). During the 3 h of perfusion, a 1.5 ml aliquot of perfusate was removed every 30 min, and 1.5 ml of fresh medium containing erythrocytes was added back to the recirculation system. At the end of the 3 h period, all 10 ml of perfusate was collected. The fi nal perfusate and all time point samples were centrifuged at 4°C to separate the medium from the erythrocytes. The lipids from each aliquot of perfusate were extracted, solubilized in 0.1% Triton X-100, and enzymatically quantifi ed. Secretion rates of lipoprotein lipids, including TG, FC, and CE, were calculated using the amount of lipid in each time point sample. To adjust for one individual animal that was a true outlier, all secretion rate data were multiplied by a factor of 10 and then log transformed.

Measurement of TG hydrolase activity
TG hydrolase activity was measured according to the methods of Schweiger et al. ( 31 ) using a radiolabeled LD substrate isolated from HepG2 cells. To prepare LD-TG substrate, HepG2 cells were grown in DMEM with 10% FBS until ‫ف‬ 70% confl uent. Cells were treated with 2.5 µCi 3 H-9,10-oleic acid/ml + 0.8 mM cold oleic acid complexed to BSA for 16 h. LDs were isolated according to the published protocol ( 32 ). Cells were washed twice with ice-cold PBS and scraped in 5 ml ice-cold PBS/150 mm dish. The contents of two dishes were combined in one 15-ml Falcon tube and centrifuged (1,000 g at 4°C, 10 min). The supernatant was removed and the cells were resuspended in 1 ml ice-cold hypotonic lysis medium (HLM) (20 mM Tris-HCl, pH 7.4, 1 mM EDTA, 10 mM sodium fl uoride, with protease inhibitor cocktail) with gentle pipetting. Resuspended cells were transferred to a Potter-Elvehjem tissue homogenizer and gently homogenized on ice. Homogenate was transferred to a clean 15 ml tube and centrifuged (1,000 g at 4°C, 10 min). The supernatant and fl oating fat layer were collected and adjusted to a fi nal concentration of 20% sucrose in HLM. This was layered into a 13.2-ml ultracentrifuge tube and gently overlaid with 5 ml ice-cold HLM containing 5% sucrose. The remainder of the tube was fi lled with HLM and centrifuged 28,000 g at 4°C for 30 min using an SW41 rotor. LDs were collected as a white band from the top of the tubes and concentrated by centrifugation (20,000 g at 4°C, 15 min). The underlying solution was removed and LDs were resuspended in Buffer A (50 mM potassium phosphate, pH 7.4, 100 mM KCl, 1 mM EDTA, plus protease inhibitor cocktail) by brief sonication. TG content of LDs was determined using commercial reagents (Roche). Once LD substrate was prepared, livers were removed and washed in ice-cold PBS containing 2 U/ml heparin and 1 mM EDTA. Liver samples ( ‫ف‬ 50 mg) were homogenized in Buffer B [20 mM Tris-HCl (pH 7.5), 0.25 M sucrose, 1 mM EDTA, 1 mM DTT, with protease inhibitor cocktail]. Cellular debris was removed by centrifugation (2,000 g at 4°C, 10 min). Tissue homogenate (100 µg) was incubated with 25 nmol 3 H-9,10-oleate labeled LD substrate and 5% defatted BSA in a total volume of 200 µl for 1 h in a 37°C water bath. Blank reactions contained no enzyme source. After incubation, the reaction was terminated by addition of 3.25 ml of methanol/chloroform/heptane (10:9:7) and 1 ml of 0.1 M potassium carbonate, 0.1 M boric acid, (pH 10.5). After centrifugation (800 g , 15 min), the release of FFA was determined by liquid scintillation counting of 1 ml of the upper phase. qPCR RNA extraction and quantitative real-time PCR (qPCR) was conducted as previously described ( 33 )   dylglycerol (PG) species. CGI-58 knockdown caused a 10-fold increase in total hepatic PG levels ( Fig. 3H ), with the majority of this increase being in atypical hepatic PG species (i.e., species other than palmitoyl-oleoyl-PG, POPG) (supplementary Fig. X). PG is usually a precursor for the synthesis of cardiolipin. Although CGI-58 ASO treatment did not signifi cantly alter the total hepatic level of cardiolipin ( Fig. 3I ), it actually decreased the major species of hepatic cardiolipin (supplementary Fig. XI). Collectively, these in vivo data support a critical role of CGI-58 in maintaining normal hepatic PL homeostasis.

CGI-58 regulates hepatic lipid secretion and fasting-induced ketogenesis
We have previously demonstrated that CGI-58-driven TG hydrolysis is critical for promoting both VLDL-TG secretion and fatty acid ␤ -oxidation in hepatoma cells ( 10 ). To investigate the role of CGI-58 in VLDL-TG secretion in vivo, we performed isolated recirculating liver perfusions and determined the hepatic secretion rates of TG, CE, and FC. ASO-mediated knockdown of CGI-58 resulted in a signifi cant reduction in the hepatic secretion rates for both TG and CE yet hepatic FC secretion was not affected by CGI-58 knockdown ( Fig. 4A ). Interestingly, CGI-58 knockdown did not alter plasma TG levels in chow-fed mice (supplementary Fig. II) and only modestly decreased postprandial plasma TG levels in HFD-fed mice ( Fig. 4B ). CGI-58 ASO treatment signifi cantly elevated total plasma cholesterol (TPC), low density lipoprotein cholesterol, and high density lipoprotein cholesterol in chow-fed mice (supplementary Figs. XII, XIII), but did not alter plasma accumulation and resulted in severe steatosis composed of both small and large fat vacuoles (mixed macrovesicular and microvesicular steatosis). A modest (2-to 3-fold) elevation in plasma alanine aminotransferase and aspartate aminotransferase levels was associated with severe steatosis in both chow-fed CGI-58 ␤ ASO-treated mice and HFD-fed CGI-58 ␤ ASO-treated mice ( Fig. 2G ); however, no infl ammatory infi ltration or fi brosis was apparent on routine hematoxylin and eosin stained liver sections at this stage of disease under our dietary conditions. Regardless of diet, CGI-58 knockdown resulted in a 4-fold increase in total hepatic TG levels ( Fig. 2H ), but there were no differences in the fatty acid species of hepatic TG between control and CGI-58 ASO-treated mice (supplementary Fig. II). Interestingly, the chow-fed CGI-58 ASO-treated mice and the HFD-fed control ASO-treated mice had similar mass levels of hepatic TG ( Fig. 2H ), yet the morphology of the LDs within those respective livers was markedly different ( Fig.  2F ). CGI-58 knockdown also resulted in signifi cantly elevated levels of hepatic diacylglycerols ( Fig. 2I ) and ceramides ( Fig. 2J ) with most molecular species of these complex lipids being elevated (supplementary Figs. III, IV). Consistent with reduced hepatic NEFA ( Fig. 2D ), total hepatic LCCoAs were signifi cantly reduced by CGI-58 ASO treatment in HFD-fed mice ( Fig. 2K ) with signifi cant reductions in monounsaturated (C16:1 and C18:1) LCCoAs (supplementary Fig. V). Collectively, these data demonstrate that CGI-58 knockdown promotes the hepatic accumulation of TG, diacylglycerols, and ceramides, while limiting the amount of hepatic NEFA and LCCoAs.

ASO-mediated knockdown of CGI-58 alters hepatic PL metabolism
Recent in vitro evidence suggests that CGI-58 is an acyl-CoA-dependent LPAAT ( 21,22 ), but the importance of CGI-58 in maintaining PL homeostasis in vivo has never been reported. We found that CGI-58 knockdown did not alter total PL levels in chow-fed mice but signifi cantly increased total hepatic PL levels in HFD-fed mice ( Fig. 3A ). Importantly, CGI-58 ASO treatment in HFD-fed mice did not alter total hepatic PA levels but did result in a significant increase in 18:0/18:1 PA ( Fig. 3C ). In addition, CGI-58 knockdown resulted in signifi cantly reduced levels of total hepatic phosphatidylcholine ( Fig. 3D ), with some molecular species of phosphatidylcholine being decreased and some being increased with CGI-58 ASO treatment (supplementary Fig. VI). Likewise, knockdown of CGI-58 caused signifi cant reductions in total hepatic phosphatidylethanolamine levels ( Fig. 3E ) with an interesting pattern of acyl-chain specifi city (supplementary Fig. VII). In contrast, CGI-58 did not alter total hepatic phosphatidylserine levels ( Fig. 3F ), yet did cause a signifi cant increase in 18:0/22:5 and 18:0/22:4 phosphatidylserine species (supplementary Fig. VIII). CGI-58 knockdown also promoted a signifi cant increase in total hepatic sphingomyelin levels ( Fig. 3G ), with the majority of the increase coming from N16:0 and N24:1 sphingomyelin species (supplementary Fig. IX). The most striking effect of CGI-58 knockdown on hepatic PL was apparent in phosphati-

CGI-58 knockdown alters gene expression in liver and white adipose tissue
To gain insights into the molecular basis of hepatic steatosis and obesity prevention driven by CGI-58 ASO treatment, we used pooled RNA samples and measured the mRNA abundance of key enzymes in TG, fatty acid, and glucose metabolism in liver and white fat, two tissues showing the most dramatic knockdown of CGI-58 expression after ASO injections ( Figs. 1, 2 ).The pooled RNA samples (n = 5 in each group) were chosen because we wanted to screen as many genes in the same metabolic pathway as possible. The pooled RNA samples have been used to show consistent changes (in the same direction) of key genes in a given metabolic or signaling pathway and these changes have been demonstrated to be physiologically relevant ( 33,(35)(36)(37)(38).
In the epididymal fat, CGI-58 knockdown by CGI-58 ␤ ASO reduced the abundance of mRNAs for the lipolytic enzymes ATGL and lipoprotein lipase (LPL), lipogenic proteins SCD-1, FAS, and SREBP-1c, and the glucose transporter 4 (GLUT4) in both chow-and HFD-fed mice ( Table  2 ). Compared with chow diet, HFD appeared to inhibit the expression of all of these genes in the epididymal fat ( Table 2 ). These data together suggest that substantial transcriptional reprogramming occurs in the liver and white adipose tissue under conditions where CGI-58 levels are diminished. Considering that CGI-58 knockdown reduced TG hydrolase cholesterol levels in HFD-fed mice ( Fig. 4D ). CGI-58 knockdown in chow-fed mice did not signifi cantly alter fed or fasting plasma NEFA levels (supplementary Fig. XII), but caused a ‫ف‬ 35% decrease in plasma NEFA levels in both the fed and fasted (12 h) state in HFD-fed mice ( Fig.  4C ). Furthermore, the levels of plasma ␤ -hydroxybutyrate following a 12 h fast were reduced by 53% in HFD-fed mice treated with CGI-58 ASO ( Fig. 4E ).

CGI-58 ASO-treated mice are protected against HFD-induced insulin resistance
Given the striking hepatic steatosis seen with CGI-58 ASO treatment ( Fig. 2 ), we anticipated that mice with CGI-58 knockdown would present with hepatic insulin resistance. To our surprise, CGI-58 ASO treatment actually prevented HFD-induced hyperglycemia ( Fig. 5A ). In HFDfed mice, CGI-58 knockdown signifi cantly reduced plasma insulin levels in both the fed and fasted state ( Fig. 5B ). Furthermore, mice treated with CGI-58 ASOs had improved glucose tolerance when fed a chow diet, and were completely protected against HFD-induced glucose intolerance ( Fig. 5C ). During glucose tolerance tests, plasma insulin levels were markedly lower in CGI-58 ASO-treated mice at all time points ( Fig. 5D ). When intraperitoneal insulin tolerance tests were performed, it was found that CGI-58 knockdown protected against HFD-induced systemic insulin resistance ( Fig. 5E ). During TG hydrolysis, TG is fi rst converted to diacylglycerols. Inhibiting CGI-58-driven TG hydrolysis may reduce the cellular diacylglycerol pool. However, the opposite was found in CGI-58 ASO-treated mice ( Fig. 2I ). Diacylglycerol can be hydrolyzed to monoacylglycerols by other lipases, synthesized from and recycled to PL, or reesterifi ed to TG. It is unclear if CGI-58 modulates hepatic diacylglycerol hydrolase activity, but it was shown that the recycling of acylglycerols to PL is defective in fi broblasts obtained from one CDS patient ( 8 ). We found that the hepatic mRNA level of acyl-CoA:diacylglycerol acyltransferase 2, an enzyme critical in diacylglycerol acylation to TG, is ‫ف‬ 50% lower in CGI-58 ASO-treated mice compared with controls ( Table 1 ). Therefore, CGI-58 inhibition may cause hepatic diacylglycerol accumulation by collectively infl uencing all these metabolic pathways directly or indirectly. Alternatively, elevated hepatic diacylglycerols and perhaps ceramides may be attributable to simple sequestration of these lipids in cytosolic LDs accumulated in the CGI-58 knockdown liver ( Fig. 2F ).
CGI-58 has an intrinsic LPAAT activity, capable of converting lysophosphatidic acid to PA in vitro ( 21,22 ). PA can be used as a precursor for the synthesis of acylglycerols and phospholipids. However, CGI-58-derived PA is unlikely used directly for, or to signifi cantly contribute to, acylglycerol synthesis because inhibiting CGI-58 increases, rather than reduces, cellular TG and diacylglycerol levels. Interestingly, ASO-mediated knockdown of CGI-58 in mice raises the hepatic PG content by ‫ف‬ 10-fold in HFD-fed mice ( Fig. 3H ). Future studies are required to defi ne how PG is accumulated in the liver of CGI-58 ASO-treated mice and whether PG accumulation is a common feature of fatty liver induced by TG hydrolysis inhibition. Additionally, PG is the precursor of cardiolipin and both lipids are enriched in mitochondria ( 40 ). Some PG species have anti-infl ammatory activity ( Fig. 2C ), our data may also suggest a feedback suppression of de novo lipogenesis when TG hydrolysis is inhibited in both liver and white adipose tissue, two important lipogenic tissues. Attenuated lipogenesis in the white adipose tissue may in part explain why mice treated with CGI-58 ASOs are resistant to HFD-induced obesity.

DISCUSSION
To our knowledge, this is the fi rst study to document the role of CGI-58 in regulating integrative lipid and glucose metabolism in adult mice. The major fi ndings of this work demonstrate that ASO-mediated knockdown of CGI-58: 1 ) prevents HFD-induced obesity and even reduces the epididymal fat pad weight in chow-fed mice, 2 ) promotes hepatic accumulation of TG, diacylglycerols, and ceramides, 3 ) alters hepatic glycerophospholipid metabolism, 4 ) diminishes hepatic contents of NEFA and LCCoAs, 5 ) decreases hepatic secretion of VLDL-TG and VLDL-CE, 6 ) blunts fasting-induced ketogenesis, and 7 ) prevents HFDinduced systemic insulin resistance.
CGI-58 interacts with ATGL to augment in vitro TG hydrolase activity of ATGL ( 12,14,16,17,39 ). Liver expresses several TG hydrolases including ATGL. Although CGI-58's lipase targets in the liver remain to be identifi ed, CGI-58 knockdown signifi cantly reduces hepatic TG hydrolase activity ( Fig. 2C ), which can at least partly explain increased hepatic TG accumulation ( Fig. 2H ). It is unlikely that hepatic steatosis seen in CGI-58 ASO-treated mice results from increased de novo lipogenesis because hepatic expression levels of all the lipogenic genes examined were substantially lower in these animals ( Table 1 ). Perhaps TG hydrolysis inhibition is sensed by cells, generating signals capable of inhibiting expression of genes related to fatty acid and TG synthesis. were downregulated with CGI-58 knockdown in the liver (Table 1). PPAR ␣ is a well-known master regulator of fatty acid oxidation and ketogenesis in the liver. Therefore, it is tempting to speculate that CGI-58-driven TG hydrolysis may liberate endogenous PPAR ␣ ligand(s) from the cytosolic LD. Nonetheless, reduced hepatic VLDL-TG secretion and fatty acid oxidation may in turn exacerbate hepatic steatosis in CGI-58 ASO-treated mice.
properties ( 41 ). In the future, it would be interesting to determine if CGI-58 inhibition infl uences fatty liver development and energy metabolism by modulating mitochondrial function and infl ammatory signaling.
It was previously demonstrated that CGI-58-driven TG hydrolysis is critical for VLDL-TG secretion in hepatoma cells ( 10,15 ). The in vivo knockdown of CGI-58 with ASOs confi rms these cell-based fi ndings ( Fig. 4A ). It is likely that CGI-58-mediated TG hydrolysis simply provides the necessary cytosolic NEFA and acylglycerol substrates for subsequent reesterifi cation in the endoplasmic reticulum for incorporation into VLDL particles, but it remains possible that CGI-58 plays other roles in VLDL assembly. We have also previously demonstrated that CGI-58-driven TG hydrolysis is coupled in increased fatty acid oxidation in cultured cells ( 10 ). Although we did not directly measure fatty acid oxidation in these studies, CGI-58 knockdown did result in diminished fasting-induced ketogenesis ( Fig. 4E ), which is primarily driven by ␤ -oxidation in the liver. In this case, it is reasonable to assume that CGI-58-mediated TG hydrolysis simply provides the necessary NEFA substrate for subsequent conversion into ketone bodies in the liver. However, it is also important to note a number of PPAR ␣ target genes Liver tissues were collected from male C57BL/6 mice treated with control (Con.) ASO or CGI-58 ␤ ASO (CGI ASO) for 8 weeks and the total RNA was extracted from individual liver samples. An equal amount of total RNA from each sample in each group (n = 5) was pooled and subjected to qPCR as described in Materials and Methods. LCAD, long-chain acyl-CoA dehydrogenase; ME, malic enzyme. Epididymal fat tissues were collected from male C57BL/6 mice treated with control (Con.) ASO or CGI-58 ␤ ASO (CGI ASO) for 8 weeks and the total RNA was extracted from individual fat samples. An equal amount of total RNA from each sample in each group (n = 5) was pooled and subjected to qPCR as described in Materials and Methods.
In summary, we have demonstrated that ASO-mediated knockdown of CGI-58 results in marked hepatic accumulation of TG, diacylglycerols, and ceramides, yet protects mice from HFD-induced obesity and systemic insulin resistance. Further investigation into the underlying molecular mechanisms should provide novel mechanistic insights into the causal relationship between lipid metabolism and insulin resistance. Given CGI-58's ubiquitous expression pattern ( 10 ) and total body knockouts die prematurely ( 11 ), tissuespecifi c knockouts will be required for these mechanistic studies in this complicated metabolic phenotype.
If CGI-58's sole function is to coactivate ATGL, one would assume that CGI-58 knockdown promotes weight gain by inhibiting fat lipolysis because ATGL knockout mice exhibit mild obesity ( 34 ). But the opposite was found and ASO-mediated knockdown of CGI-58 actually prevents HFD-induced obesity and even reduces the epididymal fat pad weight in chow-fed mice ( Fig. 1 ). In humans, mutations in CGI-58 cause TG accumulation in most tissues but fat (ectopic fat deposition) ( 1 ). Thus, our observation in CGI-58 ASO-treated mice is consistent with fi ndings in human CDS patients. It is diffi cult to measure the white adipose tissue weight in the whole-body CGI-58 knockout mice because these animals die within 16 h after birth ( 11 ). The systemic fat accumulation found in the carcass of these neonates may mainly be attributable to fat deposition in nonadipose tissues such as liver and skin ( 11 ). Nonetheless, our observation suggests that although one function of CGI-58 is to coactivate ATGL, there are likely ATGL-independent functions of CGI-58 that need to be further explored. For instance, CGI-58 was shown to activate lipases distinct from ATGL in skin ( 11 ). Thus, CGI-58 may have distinctive lipase targets in different tissues. In addition, the mRNA levels of genes related to TG hydrolysis, lipogenesis, and glucose transport are substantially lower in the white fat of mice treated with CGI-58 ASO ( Table 2 ), implying that CGI-58 ASO-treated mice may have a defect in diet-induced adipogenesis. Furthermore, CGI-58 knockdown in mice may protect against dietinduced weight gain by increasing energy expenditure because the food intake was not altered (data not shown). Future studies should focus on metabolic phenotyping of CGI-58 knockdown animals.
Hepatic steatosis is often accompanied by insulin resistance. In HFD-fed CGI-58 ASO-treated mice, however, despite severe hepatosteatosis, systemic insulin sensitivity is signifi cantly higher. Although this may be attributable to reduced weight gain in these animals, the ultimate mechanism by which CGI-58 knockdown protects against HFD-induced insulin resistance is still elusive. In CGI-58 ASO-treated mice, the hepatic diacylglycerol content was increased ( Fig.  2I ), and it has been previously demonstrated that elevated hepatic diacylglycerol levels contribute to HFD-and obesitylinked insulin resistance ( 25,26,(42)(43)(44)(45)(46). Thus the improved systemic insulin sensitivity seen in CGI-58 ASO-treated mice may be largely attributable to metabolic alterations in nonhepatic tissues. Given that CGI-58 modulates TG hydrolysis and glycerophopholipid metabolism ( Fig. 4 ) ( 21,22 ), it is also tempting to speculate that CGI-58 knockdown may improve insulin sensitivity by inhibiting generation of signaling-competent lipotoxic lipids from cytosolic LDs ( 47 ). Alternatively, CGI-58 inhibition may improve insulin sensitivity by causing sequestration of insulin signalingsuppressing lipids in cytosolic LDs away from the insulin signaling regulatory pool. This lipid compartmentalization may also explain why not all subjects or animals with hepatic steatosis develop insulin resistance. Perhaps how and where insulin signaling-suppressing lipids are accumulated is more important than the total cellular lipids in determining insulin sensitivity.